N-Acylethanolamine-hydrolyzing acid amidase (NAAA) is a lysosomal enzyme that has an important role in the deactivation of N-acylethanolamines (NAEs), bioactive lipid mediators/signaling molecules present in mammalian tissues (1, 2). In this application, we propose to develop novel NAAA inhibitors to serve as a basis for the future design of pharmacological probes and therapeutic medications. The primary substrate is N- palmitoylethanolamine (PEA), an agonist for the peroxisome proliferator-activated receptor-? (PPAR-ligand ?) (3, 4). However, there is significant activity against other NAEs including the endogenous agonists of the cannabinoid receptors CB1 and CB2, also known as endocannabinoids(5). The potential of NAAA as a druggable target has been demonstrated preclinically for analgesia in treating chronic pain and inflammation with the possibility of few or no side effects (6, 7). A reduction in the reinforcing addictive nature for drugs of abuse (8-10) has also been reported for inhibition of NAAA. These characteristics make NAAA an excellent therapeutic target for discovery of novel compounds to treat pain and inflammation without the addictive properties of opioids. In addition NAAA inhibitors are potentially important pharmacological probes by which the influence of PPAR-? signaling on addiction may be studied. At present, there are few NAAA inhibitors available, with the most successful of those published exhibiting a very short duration of action. We have developed a fluorescence-based assay through which we have screened our library of compounds and found several lead compounds that have distinct NAAA inhibitory profiles. In addition we have cloned, expressed and purified milligram amounts of NAAA and obtained the first NMR spectrum of the enzyme. With these tools in hand, we will utilize our lead compounds to probe the molecular features involved in the catalytic site of the enzyme, using a combined iophysical/biochemical approach that will elaborate structural details to inform the synthesis of next-generation NAAA-specific inhibitors. Such inhibitors will be used as probes to exploit its potential as a novel therapeutic target.